Antibody fc variants for increased blood half-life

ABSTRACT

The present invention relates to a polypeptide including an Fc variant produced by substituting a portion of the amino acid sequence of the Fc domain of a human antibody with a different amino acid sequence. The present invention also relates to an antibody including the polypeptide. The Fc variant can find application in a wide range of antibodies and Fc-fusion constructs. In one aspect, the antibody or Fc fusion construct of the present invention is a therapeutic, diagnostic or laboratory reagent, preferably a therapeutic reagent. The Fc variant is suitable for use in the treatment of cancer because its in vivo half-life can be maximized by optimization of the portion of the amino acid sequence. The antibody or Fc fusion construct of the present invention is used to kill target cells that bear a target antigen, for example cancer cells. Alternatively, the antibody or Fc fusion construct of the present invention is used to block, antagonize or agonize a target antigen. For example, the antibody or Fc fusion construct of the present invention may be used to antagonize a cytokine or a cytokine receptor.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 16/603,273, filed on Oct. 7, 2019, which is a § 371national stage entry of International Application No. PCT/KR2018/004104,filed on Apr. 6, 2018, which claims priority to Korean PatentApplication No. 10-2017-0045142, filed on Apr. 7, 2017, Korean PatentApplication No. 10-2017-0047821, filed on Apr. 13, 2017, and KoreanPatent Application No. 10-2017-0047822, filed on Apr. 13, 2017, theentire contents of which are incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(G1035-15302_SequenceListing.xml; Size: 55,226 bytes; and Date ofCreation: Aug. 5, 2022) is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to novel antibody Fc variants withincreased blood half-life and methods for producing the antibody Fcvariants.

BACKGROUND ART

With recent advances in biotechnology such as genetic recombination andcell culture, a great deal of research has been conducted on thestructure and function of proteins throughout the world. Biotechnologypromotes a better understanding of vital phenomena and plays a decisiverole in elucidating the mechanism of pathogenesis of various diseases topave the way for effective diagnosis and treatment of diseases, greatlycontributing to an improvement in the quality of life. Particularly,since hybridoma technology for monoclonal antibody production by fusingB cells with myeloma cells was developed in 1975 (Kohler and Milstein,Nature, 256:495-497, 1975), extensive research and development has beenconducted on immunotherapy using therapeutic antibodies in clinicalapplications, including cancer, autoimmune disease, inflammation,cardiovascular disease, and infection.

Therapeutic antibodies have much higher specificity for targets, areless biotoxic, and cause fewer side effects than existing small-moleculedrugs. Another advantage of therapeutic antibodies is their long bloodhalf-life (about 3 weeks). Due to these advantages, therapeuticantibodies are considered the most effective approaches for cancertreatment. Indeed, large pharmaceutical companies and researchinstitutes have concentrated their R&D capabilities on therapeuticantibodies that specifically bind to and effectively remove cancercells, including carcinogenic factors. Roche, Amgen, Johnson & Johnson,Abbott, and BMS are major pharmaceutical companies that are currentlydeveloping therapeutic antibody drugs. Particularly, Roche, whodeveloped three innovative therapeutic antibodies, i.e. Herceptin,Avastin, and Rituxan, for applications in anticancer therapy, reachedapproximately 19.5 billion US dollars in sales for the therapeuticantibodies in 2012 to gain huge profits in the global market and iscurrently leading the global market for antibody drugs. Johnson &Johnson, who developed Remicade, is rapidly growing in the global marketfor antibodies due to the increased sales volume of Remicade. Otherpharmaceutical companies such as Abbott and BMS are known to possessmany therapeutic antibodies in the final stage of development. As aconsequence, biomedicines, including therapeutic antibodies that arespecific for target diseases and cause few side effects, are rapidlyreplacing small-molecule medicines that have led the globalpharmaceutical market.

The Fc region of an antibody recruits immune leukocytes or serumcomplement molecules, which in turn triggers the clearance of defectivecells such as tumor cells or infected cells. The Fc interface betweenCγ2 and Cγ3 domains mediates interactions with the neonatal Fc receptor(FcRn) and its binding recycles endocytosed antibody from the endosomeback to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). Thisprocess, coupled with preclusion of kidney filtration due to the largesize of the full-length IgG antibody molecule, results in favorableantibody serum half-life in the range of 1-3 weeks. Further, binding ofFc to FcRn plays a key role in antibody transport. Accordingly, the Fcregion is crucial for the prolonged serum persistence of circulatingantibodies through an intracellular trafficking and recycle mechanism.

The administration of an antibody or an Fc-fusion protein as atherapeutic agent requires a predetermined frequency of injection takinginto consideration the half-life of the therapeutic agent. A longer invivo half-life allows for less frequent injection or lower dosing. Thus,in many clinical studies that are currently underway, many efforts haveconcentrated on the development of next-generation anticancertherapeutic antibodies and anticancer therapeutic proteins by theintroduction of mutations into the Fc domain to increase the half-lifeof antibodies or the introduction of variants into the Fc domain toachieve a maximal ADCC effect (Modified from Cancer Immunol Res.2015/Thomson Reuters).

However, despite the research groups' efforts aimed at developing someproteins and antibodies with increased binding affinity for FcRn andextended in vivo half-life by introducing some mutations into the Fcdomain, a significant increase in in vivo half-life is not yet achieved.Under these circumstances, there is an urgent need to develop optimallymutated antibodies.

The description of the Background Art is merely provided for betterunderstanding the background of the invention and should not be taken ascorresponding to the prior art already known to those skilled in theart.

DETAILED DESCRIPTION OF THE INVENTION Problems to be Solved by theInvention

The present inventors have earnestly conducted research to efficientlyincrease the in vivo half-life of existing therapeutic proteins orantibodies, and as a result, found that a therapeutic protein orantibody is optimized by substituting a portion of the amino acidsequence of the wild-type Fc domain with a different amino acid sequenceso that its blood half-life can be maximized while maintaining itssuperior activity.

One object of the present invention is to provide a polypeptideincluding an Fc variant produced by substituting a portion of the aminoacid sequence of the Fc domain of a human antibody with a differentamino acid sequence.

A further object of the present invention is to provide an antibodyincluding the polypeptide.

Another object of the present invention is to provide a nucleic acidmolecule encoding the polypeptide.

Another object of the present invention is to provide a vector includingthe nucleic acid molecule.

Another object of the present invention is to provide a host cellincluding the vector.

Another object of the present invention is to provide a compositionincluding the polypeptide, the antibody, the nucleic acid molecule orthe vector.

Another object of the present invention is to provide a method forproducing the polypeptide or the antibody.

Still another object of the present invention is to provide a method forscreening the polypeptide.

Other objects and advantages of the invention become more apparent fromthe following detailed description, claims, and drawings.

Means for Solving the Problems

One aspect of the present invention provides a polypeptide including anFc variant produced by substituting a portion of the amino acid sequenceof the Fc domain of a human antibody with a different amino acidsequence.

A further aspect of the present invention provides a composition forincreasing the blood half-life of a therapeutic antibody or protein,including an Fc variant produced by substituting a portion of the aminoacid sequence of the Fc domain of a human antibody with a differentamino acid sequence.

The present inventors tried to find an approach for efficientlyincreasing the in vivo half-life of an existing therapeutic protein orantibody, and as a result, found that a therapeutic protein or antibodyincluding an FC variant produced by substituting and optimizing aportion of the amino acid sequence of the wild-type Fc domain with adifferent amino acid sequence can achieve a maximum in vivo half-life.

An antibody is a protein that specifically binds to a specific antigen.A natural antibody is a heterodimeric glycoprotein with a molecularweight of about 150,000 daltons that usually consists of two identicallight chains (L) and two identical heavy chains (H).

The human antibody used in the present invention belongs to one of thefive major classes: IgA, IgD, IgE, IgG, and IgM. The human antibody ispreferably an IgG antibody. Papain digestion of antibodies procures twoFab fragments and one Fc fragment, and the Fc region of a human IgGmolecule is generated by papain digestion of the N-terminus of Cys 226(Deisenhofer, Biochemistry 20: 2361-2370, 1981).

The antibody Fc domain may be the Fc domain of an IgA, IgM, IgE, IgD orIgG antibody or its modifications. In one embodiment, the domain is theFc domain of an IgG antibody, for example, an IgG1, IgG2a, IgG2b, IgG3or IgG4 antibody. In one embodiment, the Fc domain may be an IgG1 Fcdomain, for example, the Fc domain of an anti-HER2 antibody, preferablythe Fc domain of trastuzumab, more preferably the Fc domain having thesequence set forth in SEQ ID NO: 28. The polypeptide of the presentinvention may be optionally partially or fully glycosylated. Thepolypeptide of the present invention may further include one or moreregions derived from the antibody in addition to the Fc domain. Inaddition, the polypeptide of the present invention may include anantigen binding domain derived from the antibody and may form anantibody or antibody-like protein with another polypeptide.

Herein, the amino acid residues of the antibody Fc domain are designatedaccording to the Kabat EU numbering system usually used in the art, asdescribed in Kabat et al., “Sequences of Proteins of ImmunologicalInterest” 5th Ed., U.S. Department of Health and Human Services, NIHPublication No. 91-3242, 1991).

According to a preferred embodiment of the present invention, thesubstituted Fc variant includes, as an amino acid substitution, M428Laccording to the Kabat EU numbering system.

According to a preferred embodiment of the present invention, thesubstituted Fc variant includes, as amino acid substitutions, a) M428Land b) Q311R or L309G according to the Kabat EU numbering system.

According to a preferred embodiment of the present invention, thesubstituted Fc variant includes, as amino acid substitutions, P228L andM428L according to the Kabat EU numbering system.

According to a preferred embodiment of the present invention, the Fcvariant including the amino acid substitutions P228L and M428L includesadditional amino acid substitutions at one or more positions selectedfrom the group consisting of positions 234, 264, 269, 292, 309, 342,359, 364, 368, 388, 394, 422, 434, and 445 according to the Kabat EUnumbering system.

The additional amino acid substitutions may be L309R and N434S.

The additional amino acid substitutions may be V264M, L368Q, E388D,V422D, and P445S.

The additional amino acid substitutions may be R292L, T359A, and S364G.

The additional amino acid substitutions may be L234F, E269D, Q342L,E388D, and T394A.

According to a preferred embodiment of the present invention, thesubstituted Fc variant includes, as amino acid substitutions, a) M428Land b) P230Q or P230S according to the Kabat EU numbering system.

According to a preferred embodiment of the present invention, thesubstituted Fc variant including the amino acid substitutions a) M428Land b) P230Q or P230S includes additional amino acid substitutions atone or more positions selected from the group consisting of positions243, 246, 295, 320, 356, 361, 384, and 405 according to Kabat EUnumbering system.

The additional amino acid substitutions may be F243Y, K246E, N361S, andN384I.

The additional amino acid substitutions may be Q295L, K320M, D356E, andF405I.

The present invention is directed to an Fc variant including one or moreamino acid substitutions that regulate its binding to and dissociationfrom the neonatal Fc receptor (FcRn). Particularly, the Fc variant ofthe present invention or its functional variant exhibits increasedbinding affinity for FcRn under acidic conditions (at a pH lower than 7)and very low binding force to FcRn under neutral pH conditions.

The therapeutic antibody whose half-life is to be increased is notparticularly limited and examples thereof include polyclonal antibodies,monoclonal antibodies, minibodies, domain antibodies, bispecificantibodies, antibody mimetics, chimeric antibodies, antibody conjugates,human antibodies, humanized antibodies, and their fragments.

As the monoclonal antibodies, there may be mentioned, for example: humanantibodies, such as panitumumab (Vectibix), ofatumumab (Arzerra),golimumab (Simponi), and ipilimumab (Yervoy); humanized antibodies, suchas tocilizumab (Actemra), trastuzumab (Herceptin), bevacizumab(Avastin), omalizumab (Xolair), mepolizumab (Bosatria), gemtuzumabozogamicin (Mylotarg), palivizumab (Synagis), ranibizumab (Lucentis),certolizumab (Cimzia), ocrelizumab, mogamulizumab (Poteligeo), andeculizumab (Soliris); and chimeric antibodies, such as rituximab(Rituxan), cetuximab (Erbitux), infliximab (Remicade), and basiliximab(Simulect).

The therapeutic protein whose half-life is to be increased is notparticularly limited and examples thereof include: hormones, such asinsulin; cytokines, such as growth factors, interferons, interleukins,erythropoietin, neutrophil growth factors, and transforming growthfactors; Fc fusion proteins, such as etanercept (Enbrel), aflibercept(Eylea, Zaltrap), abatacept (Orencia), alefacept (Amevive), belatacept(Nulojix), and rilonacept (Arcalyst); therapeutic peptides, such asteriparatide (Forteo), exenatide (Byetta), liraglutide (Victoza),lanreotide (Somatuline), pramlintide (Symlin), and enfuvirtide (Fuzeon);and polypeptides including, in part or in whole, VEGF receptors, Her2receptors, G-protein-coupled receptors, and cell surface receptors ofion channels.

The half-life of the therapeutic antibody or protein can be extended bybinding to the polypeptide of the present invention or a nucleic acidencoding the polypeptide or introducing into a vector expressing thenucleic acid.

According to a preferred embodiment of the present invention, thebinding affinity of the Fc variant for FcRn at a pH of 5.6 to 6.4(preferably 5.8 to 6.2) is higher by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100% than that of the wild-type Fcdomain or by at least 2 times, at least 3 times, at least 4 times, atleast 5 times, at least 6 times, at least 7 times, at least 8 times, atleast 9 times, at least 10 times, at least 20 times, at least 30 times,at least 40 times, at least 50 times, at least 60 times, at least 70times, at least 80 times, at least 90 times or at least 100 times thanthat of the wild-type Fc domain.

According to a preferred embodiment of the present invention, the degreeof dissociation of the Fc variant from the neonatal Fc receptor (FcRn)at a pH of 7.0 to 7.8 (preferably 7.2 to 7.6) may be the same as or notsubstantially different from that of the wild-type Fc domain.

According to one embodiment of the present invention, the substituted Fcvariant exhibits much higher binding affinity under weakly acidicconditions (for example, at a pH of 5.8 to 6.2) than the wild-type Fc orother developed Fc variants and its degree of dissociation under neutralconditions (for example, at a pH of 7.4) is the same as or substantiallyequivalent to or higher than that of the wild-type Fc or other developedFc variants (see Examples 4 and 8).

According to a preferred embodiment of the present invention, thesubstituted Fc variant has a long half-life compared to the wild type.

The half-life of the substituted Fc variant according to the presentinvention may be longer by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 100% than that of the wild-type Fc domain or atleast two times, at least 3 times, at least 4 times, at least 5 times,at least 6 times, at least 7 times, at least 8 times, at least 9 timesor at least 10 times that of the wild-type Fc domain.

According to one embodiment of the present invention, the substituted Fcvariant has a significantly improved in vivo half-life compared to thewild type (see Example 11 and Table 3).

By “Fc gamma receptor” or “FcγR” as used herein is meant any member of afamily of proteins that bind to the Fc region of an IgG antibody and areencoded by the FcγR gene. Examples of such Fc gamma receptors or FcγRsinclude, but are not limited to: FcγRI(CD64), including FcγRIa, FcγRIb,and FcγRIc; FcγRII(CD32), including FcγRIIa, FcγRIIb, and FcγRIIc;FcγRIII(CD16), including FcγRIIIa and FcγRIIIb; and undiscovered FcγRs.The FcγR may be derived from mammalian organisms, including humans,mice, rats, rabbits, and monkeys, and other organisms.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds to the Fc region of an IgG antibody and is encoded at leastpartially by the FcRn gene. The FcRn may be derived from mammalianorganisms, including humans, mice, rats, rabbits, and monkeys, and otherorganisms. The functional FcRn protein includes two polypeptides, whichare referred to as heavy and light chains. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.

Another aspect of the present invention provides an antibody includingthe polypeptide.

As used herein, the term “antibody” refers to a polyclonal antibody, amonoclonal antibody, a minibody, a domain antibody, a bispecificantibody, an antibody mimetic, a chimeric antibody, an antibodyconjugate, a human antibody, a humanized antibody or its fragment (forexample, an antigen binding antibody fragment).

According to a preferred embodiment of the present invention, thehalf-life of the Fc domain or the polypeptide including the Fc domaincan be maximized by optimization of the corresponding antibody Fcregions (for example, M428L and Q311R; or M428L and L309G).

Another aspect of the present invention provides a nucleic acid moleculeencoding the polypeptide, a vector including the nucleic acid moleculeor a host cell including the vector.

The nucleic acid molecule of the present invention may be an isolated orrecombinant nucleic acid molecule. Examples of such nucleic acidsinclude single- and double-stranded DNA and RNA and their correspondingcomplementary sequences. The isolated nucleic acid may be isolated froma naturally occurring source. In this case, the isolated nucleic acid isseparated from the peripheral gene sequence present in the genome of asubject from which the nucleic acid is to be isolated. The isolatednucleic acid may be understood as a nucleic acid, for example, a PCRproduct, a cDNA molecule or an oligonucleotide, that is enzymatically orchemically synthesized from a template. In this case, the nucleic acidproduced from this procedure can be understood as the isolated nucleicacid molecule. The isolated nucleic acid molecule represents a nucleicacid molecule in the form of a separate fragment or as a component of alarger nucleic acid construct. A nucleic acid is “operably linked” whenarranged in a functional relationship with another nucleic acidsequence. For example, the DNA of a presequence or secretory leader isoperably linked to the DNA of the polypeptide when expressed as apreprotein, which is a presecretory polypeptide. A promoter or anenhancer affecting the transcription of the polypeptide sequence isoperably linked to a coding sequence or a ribosome-binding site isoperably linked to a coding sequence when it is arranged such thattranslation is promoted. Generally, the term “operably linked” meansthat DNA sequences to be linked are located adjacent to each other. Inthe case of secretory leaders, the term “operably linked” means that thesecretory leaders are present adjacent to each other in the same leadingframe. However, an enhancer need is not necessarily contiguous. Thelinkage is performed by ligation at a convenient restriction enzymesite. In the case where this site does not exist, a syntheticoligonucleotide adaptor or a linker is used according to a suitablemethod known in the art.

As used herein, the term “vector” is used to refer to a carrier intowhich a nucleic acid sequence can be inserted for introduction into acell where it can be replicated. A nucleic acid sequence can be“exogenous,” or “heterologous”. Vectors include plasmids, cosmids andviruses (e.g., bacteriophage). One of skill in the art may construct avector through standard recombinant techniques, which are described inManiatis et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In:Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, N Y,1994).

As used herein, the term “expression vector” refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. Expressionvectors can contain a variety of “control sequences”. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well.

As used herein, the term “host cell” refers to any transgenic organismthat is capable of replicating the vector or expressing the gene encodedby the vector. Suitable organisms include eukaryotes and prokaryotes.The host cell may be transfected or transformed by the vector. Thetransfection or transformation refers to a process for transferring orintroducing the exogenous nucleic acid molecule into the host cell.

The host cell of the present invention is preferably a bacterial cell,CHO cell, HeLa cell, HEK293 cell, BHK-21 cell, COS7 cell, COPS cell,A549 cell or NIH3T3 cell, but is not limited thereto.

Another aspect of the present invention provides a method for producinga polypeptide including a human antibody Fc variant, including: a)culturing a host cell including a vector including a nucleic acidmolecule encoding the polypeptide; and b) collecting the polypeptideexpressed by the host cell.

Another aspect of the present invention provides a method for producingan antibody, including: a) culturing a host cell expressing an antibodyincluding the polypeptide; and b) purifying the antibody expressed bythe host cell.

In the method of the present invention, the antibody may be purified byfiltration, HPLC, anion exchange or cation exchange, high-performanceliquid chromatography (HPLC), affinity chromatography or a combinationthereof, preferably affinity chromatography using Protein A.

Another aspect of the present invention provides a method for screeninga polypeptide including an Fc variant, including: constructing a libraryof Fc variants including, as a mutation, M428L according to the Kabat EUnumbering system; and b) sorting an Fc variant having a higher affinityfor FcRn at a pH of 5.6 to 6.4 than the wild type from the Fc variantsincluding the M428L mutation.

The Fc variants including the M428L mutation may include at least oneadditional amino acid substitution.

According to a preferred embodiment of the present invention, theadditional amino acid substitution includes Q311R or L309G as amutation.

According to a preferred embodiment of the present invention, theadditional amino acid substitution includes P228L as a mutation.

The Fc variants including the P228L mutation may include at least oneadditional amino acid substitution.

The additional amino acid substitution is not particularly limited butis preferably an amino acid mutation at at least one position selectedfrom the group consisting of positions 234, 264, 269, 292, 309, 342,359, 364, 368, 388, 394, 422, 434, and 445 according to the Kabat EUnumbering system.

According to a preferred embodiment of the present invention, theadditional amino acid substitution includes P230Q or P230S as amutation.

The Fc variants including the P230 mutation may include at least oneadditional amino acid substitution.

The additional amino acid substitution is not particularly limited butis preferably an amino acid mutation at at least one position selectedfrom the group consisting of positions 243, 246, 295, 320, 356, 361,384, and 405 according to the Kabat EU numbering system.

The screening method of the present invention can usefluorescence-activated cell sorting (FACS) or automated flow cytometry.Instruments for flow cytometry are well known to those skilled in theart. Examples of such instruments include FACSAria, FACS Star Plus,FACScan, and FACSort (Becton Dickinson, Foster City, Calif.), Epics C(Coulter Epics Division, Hialeah, Fla.), MOFLO (Cytomation, ColoradoSprings, Colo), and MOFLO-XDP (Beckman Coulter, Indianapolis, Ind.).Flow cytometry generally involves the separation of cells or otherparticles in a liquid sample. Typically, the purpose of flow cytometryis to analyze the separated particles for one or more characteristicsthereof, for example, presence of a labeled ligand or other molecule.The particles are passed one by one by the sensor and are sorted basedon size, refraction, light scattering, opacity, roughness, shape,fluorescence, etc.

Another aspect of the present invention provides a composition includingthe polypeptide including the Fc variant including one or more aminoacid substitutions, the antibody, the nucleic acid molecule or thevector.

According to a preferred embodiment of the present invention, thecomposition is a pharmaceutical composition for preventing or treatingcancer.

According to a preferred embodiment of the present invention, thepharmaceutical composition (or the polypeptide, the antibody, thenucleic acid molecule or the vector) recognizes a cancer antigen.

According to one embodiment of the present invention, the Fc variant hasantibody dependent cellular cytotoxicity (ADCC) activity comparable orsuperior to that of a control group (for example, trastuzumab),achieving significantly increased half-life and high anticancer activity(see Example 13 and FIG. 18 ).

The pharmaceutical composition of the present invention may include (a)the polypeptide, the antibody, the nucleic acid molecule encoding thepolypeptide or the vector including the nucleic acid molecule and (b) apharmaceutically acceptable carrier.

Yet another aspect of the present invention provides a method forpreventing or treating cancer, including administering thepharmaceutical composition to a subject.

The type of the cancer to be prevented or treated by the method of thepresent invention is not limited. The pharmaceutical composition of thepresent invention can be administered to treat a number of cancers,including leukemias and lymphomas such as acute lymphocytic leukemia,acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronicmyelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas,multiple myeloma, childhood solid tumors such as brain tumors,neuroblastoma, retinoblastoma, Wilms Tumor, bone tumors, and soft-tissuesarcomas, common solid tumors of adults such as lung cancer, breastcancer, prostate cancer, urinary cancers, uterine cancers, oral cancers,pancreatic cancer, melanoma and other skin cancers, stomach cancer,ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroidcancer, esophageal cancer, and testicular cancer.

The pharmaceutically acceptable carrier of the pharmaceuticalcomposition according to the present invention may be any of those knownin the art. Examples of carriers suitable for use in the pharmaceuticalcomposition of the present invention include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia,calcium phosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and mineral oil. The pharmaceutical composition ofthe present invention may further include at least one additive selectedfrom the group consisting of lubricating agents, wetting agents,sweetening agents, flavoring agents, emulsifying agents, suspendingagents, and preservatives. Details of suitable pharmaceuticallyacceptable carriers and formulations can be found in Remington'sPharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can beadministered orally or parenterally, preferably parenterally. Forexample, the pharmaceutical composition of the present invention may beadministered by intravenous, local or intraperitoneal injection.

The subject is not particularly limited but is preferably construed toinclude vertebrates, more preferably primates, including humans andchimpanzees, household pets, including dogs and cats, livestock,including cattle, horses, sheep, and goats, and rodents, including miceand rats.

A suitable dose of the pharmaceutical composition according to thepresent invention depends on a variety of factors such as formulation,mode of administration, age, body weight, sex, and pathologicalcondition of the patient, diet, time and route of administration, rateof excretion, and responsiveness. A physician having ordinary skill inthe art can readily determine and prescribe an effective dose of thepharmaceutical composition according to the present invention for thedesired treatment or prevention. According to a preferred embodiment ofthe present invention, the daily dose of the pharmaceutical compositionaccording to the present invention is from 0.0001 to 100 mg/kg.

The pharmaceutical composition of the present invention can be preparedin unit dosage forms or dispensed in multi-dose containers with apharmaceutically acceptable carrier and/or excipient by a suitablemethod which can be easily carried out by one having ordinary skill inthe art. The pharmaceutical composition of the present invention may bein the form of a solution, suspension or emulsion in an oil or aqueousmedium. The pharmaceutical composition of the present invention may bein the form of an extract, powder, granule, tablet or capsule. Thepharmaceutical composition of the present invention may further includea dispersant or a stabilizer.

The pharmaceutical composition of the present invention can be used fora single therapy. Alternatively, the pharmaceutical composition of thepresent invention may be used in combination with general chemotherapyor radiotherapy. This combined therapy is more effective for cancertreatment. Chemotherapeutic agents that can be used with the compositionof the present invention include cisplatin, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol,transplatinum, 5-fluorouracil, vincristin, vinblastine, andmethotrexate, and the like. Radiation therapies that can be used withthe composition of the present invention include X-ray irradiation andγ-ray irradiation.

Effects of the Invention

The features and advantages of the present invention are summarized asfollows.

(i) The present invention provides a polypeptide including an Fc variantproduced by substituting a portion of the amino acid sequence of the Fcdomain of a human antibody with a different amino acid sequence.

(ii) The present invention also provides a method for producing thepolypeptide or an antibody including the polypeptide.

(iii) The Fc variant of the present invention is suitable for use in thetreatment of cancer because its in vivo half-life can be maximized byoptimization of the portion of the amino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression vectors for expression and purification oftetrameric FcRn and dimeric FcRn and SDS-PAGE gels after purification.

FIG. 2 is a schematic diagram of a library constructed such that 18amino acids are contained at positions M252 and M428.

FIG. 3 shows a 2M library search process and a sorted M428L variant.

FIG. 4 schematically shows an error library and a point libraryconstructed based on M428L.

FIGS. 5A and 5B show FACS fluorescence intensities of variants sortedfrom (FIG. 5A) an error library and (FIG. 5B) a point library.

FIG. 6 shows plasmids for expressing trastuzumab heavy and light chainsin animal cells.

FIG. 7 shows expression and purification results for wild-typetrastuzumab.

FIGS. 8A1, 8A2, and 8B compare the physical properties of commercialtrastuzumab with those of in-house trastuzumab (FIGS. 8A1 and 8A2:CE-cIEF, FIG. 8B: SEC).

FIGS. 9A and 9B compare the physical properties of commercialtrastuzumab with those of in-house trastuzumab (FIG. 9A) by N-glycanprofiling (FIG. 9B).

FIGS. 10A, 10B and 10C show expression and purification results for 10trastuzumab Fc variants (FIG. 10A: affinity chromatography, FIG. 10B:SDS-PAGE analysis, FIG. 10C: list of final yield).

FIGS. 11A and 11B show SEC characterization results for trastuzumab Fcvariants.

FIGS. 12A, 12B, 12C and 12D show binding forces of trastuzumab Fcvariants to FcRn, which were measured by ELISA.

FIGS. 13A and 13B show binding forces of trastuzumab Fc variants tohFcRn at pH values of 6.0 and 7.4, which were measured using a BiaCoreinstrument (FIG. 13A: pH 6.0 (capture method) FIG. 13B: pH 7.4 (avidformat)).

FIG. 14 compares the pharmacokinetics of commercial trastuzumab withthose of in-house trastuzumab in regular mice (C57BL/6J (B6)) and humanFcRn Tg mice.

FIG. 15 shows the results of pharmacokinetic analysis for Fc variants inhuman FcRn Tg mice (after intravenous injection of the variants (5 mg/kgeach), n=5).

FIGS. 16A thru 16L show binding forces of trastuzumab Fc variants toFcγRs, 16 a, FcγRI-PFc; FIG. 16 b , FcγRI-EFc, FIG. 16 c , FcγRIIb-PFc;FIG. 16 d , FcγRIIb-EFc, FIG. 16 e , FcγRIIa(H)-PFc; FIG. 16 f ,FcγRIIa(H)-EFc, FIG. 16 g , FcγRIIa(R)-PFc; FIG. 16 h , FcγRIIa(R)-EFc,FIG. 16 i , FcγRIIIa(V)-PFc; FIG. 16 j , FcγRIIIa-EFc, FIG. 16 k ,FcγRIIIa(F)-PFc; FIG. 16 l , FcγRIIIa(F)-EFc), which were measured byELISA.

FIG. 17 compares the effector functions of trastuzumab Fc variants withthose of normal IgG and trastuzumab as a control group (ADCC assay).

FIG. 18 compares the effector functions (ADCC) of trastuzumab Fcvariants.

FIG. 19 shows binding forces of trastuzumab Fc variants to C1q, whichwere measured by ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in more detail with reference tothe following examples. It will be evident to those skilled in the artthat the scope of the present invention is not limited by these examplesaccording to the gist of the present invention.

EXAMPLES Example 1: Expression and Purification of Neonatal Fc Receptor(FcRn) for Searching Library of Fc Variants

Tetrameric FcRn and dimeric FcRn for searching Fc variants with improvedpH-dependent binding force to FcRn were expressed and purified. To thisend, expression vectors were prepared (FIG. 1 ). pMAZ-β2microglobulin-GSlinker-FcRnα-chain-streptavidin-His was constructed as aDNA plasmid to obtain tetrameric FcRn. The DNA was co-transfected intoHEK 293F cells and temporarily expressed at a level of 300 ml. Theresulting culture medium was centrifuged at 7000 rpm for 10 min. Thecollected supernatant was equilibrated with 25×PBS and filtered with a0.2 μm bottle top filter (Merck Millipore). After equilibration withPBS, FcRn was allowed to bind to Ni-NTA resin (Qiagen) at 4° C. for 16h. The FcRn-bound resin was loaded onto a column and the column waseluted with 50 ml of wash-1 buffer (PBS), 25 ml of wash-2 buffer (PBS+10mM imidazole), 25 ml of wash-3 buffer (PBS+20 mM imidazole), and 200 μlof wash-4 buffer (PBS+250 mM imidazole) to remove proteins other thantFcRn. Then, 2.5 ml of elution buffer (PBS+250 mM imidazole) was allowedto flow through the column to obtain tFcRn. The buffer was replaced witha new one using centrifugal filter units (Merck Millipore). Dimeric FcRnwas obtained from pcDNA-FcRnα-chain-GST-β2 microglobulin plasmid, whichwas received from the University of Oslo. The DNA was co-transfectedinto HEK 293F cells and temporarily expressed at a level of 300 ml. Theresulting culture medium was centrifuged at 7000 rpm for 10 min. Thecollected supernatant was equilibrated with 25×PBS and filtered with a0.2 μm bottle top filter (Merck Millipore). After equilibration withPBS, FcRn was allowed to bind to Glutathione Agarose 4B (incospharm) at4° C. for 16 h. The FcRn-bound resin was loaded onto a column and thecolumn was eluted with 10 ml of wash buffer (PBS) to remove proteinsother than dFcRn. Then, 2.5 ml of elution buffer (50 mM Tris-HCl+10 mMGSH pH 8.0) was allowed to flow through the column. The buffer wasreplaced with a new one using centrifugal filter units 3K (MerckMillipore). The sizes of the tetrameric FcRn and dimeric FcRn afterpurification were determined using SDS-PAGE gels (FIG. 1 ). The purifiedtetrameric FcRn and dimeric FcRn were fluorescently labeled with Alexa488 for fluorescence detection.

Example 2: Construction of 2M Library of Fc Variants

pMopac12-NlpA-Fc-FLAG was constructed from the gene (SEQ ID NO: 29) ofthe Fc domain of trastuzumab using SfiI restriction enzyme. Based on thevector, library inserts were constructed using pMopac12-seq-Fw,Fc-M252-1-Rv, Fc-M252-2-Rv, Fc-M252-3-Rv, Fc-M428-Fw, Fc-M428-1-Rv,Fc-M428-2-Rv, Fc-M428-3-Rv, Fc-M428-frg3-Fw, and pMopac12-seq-Rv primerssuch that two Met residues in the Fc were substituted with 18 differentamino acids except Cys and Met (Table 1 and FIG. 2 ). The inserts weretreated with SfiI restriction enzyme and ligated with the vector treatedwith the same SfiI. Thereafter, the ligated inserts were transformedinto E. coli Jude1 ((F′[Tn10(Tet^(r))proAB⁺lacl^(q)Δ(lacZ)M15]mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dlacZΔM15 ΔlacX74 deoR recA1 araD139Δ(araleu)7697 galU galKrpsLendA1nupG) to establish a large 2M library of Fcvariants (library size: 1×10⁹).

TABLE 1 pMopac12-seq- 5′-CCAGGCTTTACACTTTATGC-3′ Ew Fc-M252-1-Rv5′-CCTCAGGGGTCCGGGAGATGWAGAGGGTGTC CTTGGGTTTTGGG-3′ Fc-M252-2-Rv5′-CCTCAGGGGTCCGGGAGATKNBGAGGGTGTC CTTGGGTTTTGGG-3′ Fc-M252-3-Rv5′-CCTCAGGGGTCCGGGAGATCCAGAGGGTGTC CTTGGGTTTTGGG-3′ Fc-M428-Fw5′-ATCTCCCGGACCCCTGAGG-3′ Fc-M429-1-Rv5′-GTAGTGGTTGTGCAGAGCCTCATGGWACACG GAGCATGAGAAGACGTTCC-3′ Fc-M428-2-Rv5′-GTAGTGGTTGTGCAGAGCCTCATGKNBCACG GAGCATGAGAAGACGTTCC-3′ Fc-M428-3-Rv5′-GTAGTGGTTGTGCAGAGCCTCATGCCACACG GAGCATGAGAAGACGTTCC-3′ Fc-M428-frg3-5′-CATGAGGCTCTGCACAACCACTAC-3′ Fw pMopac12-seq-5′-CTGCCCATGTTGACGATTG-3′ Rv FC-Sub#0-Rv 5′-GTCCTTGGGTTTTGGGGGGAAG-3′ FC-Sub#1-1-Fw 5′- CTTCCCCCCAAAACCCAAGGACNNKCTCATGATCTCCCGGACCCCTGAGGTCACATGCG-3′ Fc-Sub*1-2-Fw5′- CTTCCCCCCAAAACCCAAGGACACCNNKAT GATCTCCCGGACCCCTGAGGTCACATGCG-3′Fc-Sub*1-3-Fw 5′- CTTCCCCCCAAAACCCAAGGACACCCTCATGNNKTCCCGGACCCCTGAGGTCACATGCG-3′ Fc-Sub#1-4-Fw5′- CTTCCCCCCAAAACCCAAGGACACCCTCAT GATCNNKCGGACCCCTGAGGTCACATGCG-3′Fc-Sub#1-5-Fw 5′- CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCNNKACCCCTGAGGTCACATGCG-3′ Fc-Sub#1-Rv 5′- GACGGTGAGGAGCGCTGACC-3′Fc-Sub#2-1-Fw 5′- GGTCAGCGTCCTCACCGTCNNKCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG- 3′ Fc-Sub#2-2-Fw5′- GGTCAGCGTCCTCACCGTCCTGCACNNKGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGG- 3′Fc-Sub#2-3-Fw 5′- GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGNNKAATGGCAAGGAGTACAAGTGCAAGG- 3′ Fc-Sub#2-Rv5′- CACGGAGCATGAGAAGACGTTCC-3′ Fc-Sub#3-1-Fw5′- GGAACGTCTTCTCATGCTCCGTGCTGCATN NKGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG-3′ Fc-Sub#3-2-Fw 5′- GGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTNNKCACAACCACTACACGCAGAAGAGCCT CTCCCTG-3′ Fc-Sub#3-3-Fw5′- GGAACGTCTTCTCATGCTCCGTGCTGCATG AGGCTCTGCACNNKCACTACACGCAGAAGAGCCTCTCCCTG-3′ Fc-Sub#3-4-Fw 5′- GGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAACCACNNKACGCAGAAGAGCCT CTCCCTG-3′ ep-Fc-Fw5′- CCAGCCGGCCATGGCG-3′ ep-Fc-Rv 5′- GAATTCGGCCCCCGAGGCCCC-3′ Primersused for cloning (SEQ ID NOS: 1-27)

Example 3: Search Against the 2M Library of Fc Variants Based onBacterial Culture and Flow Cytometry

In this example, a search was conducted against the established 2Mlibrary of Fc variants. Specifically, 1 ml of Fc variant library cellstransformed into E. coli Jude 1 cells were cultured with shaking inTerrific broth (TB) medium supplemented with 2% (w/v) glucose andchloramphenicol (40 μg/mL) as an antibiotic at 37° C. and 250 rpm for 4h. After shaking culture, the library cells were inoculated into TBmedium in a ratio of 1:100 and cultured with shaking at 250 rpm and 37°C. until an OD₆₀₀ of 0.5 was reached. Thereafter, culture was furtherperformed at 25° C. for 20 min for cooling and 1 mMisopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induceexpression. After completion of the culture, the collected cells weredivided into equal amounts based on OD600 normalization, followed bycentrifugation at 14000 rpm for 1 min. The harvested cells wereresuspended in 1 ml of 10 mM Tris-HCl (pH 8.0) and washed twice bycentrifugation for 1 min. Cells were resuspended in 1 ml of STE (0.5 Msucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)) and centrifuged at 37° C.for 30 min to remove the outer membrane. The supernatant was discardedby centrifugation and 1 ml of Solution A (0.5 M sucrose, 20 mM MgCl₂, 10mM MOPS (pH 6.8)) was added, followed by resuspension andcentrifugation. Cells were resuspended in 1 ml of a mixture of 1 ml ofSolution A and 20 μl of 50 mg/ml lysozyme solution, followed bycentrifugation at 37° C. for 15 min to remove the peptidoglycan layer.The supernatant was removed and cells were resuspended in 1 ml of PBS.300 μl of the suspension was added with 700 μl of PBS and fluorescentlylabeled tetrameric FcγRIIIa-Alexa 488 fluor probe and centrifuged atroom temperature to label the fluorescent probe with spheroplast. Afterthe labeling, cells were washed once with 1 ml of PBS and sorting wasperformed by flow cytometry (S3 sortor (Bio-rad)) to collect the top 3%highly fluorescent cells. The sorted cells were resorted for higherpurity. For the resorted sample, genes were amplified by PCR using Taqpolymerase (Biosesang) with pMopac12-seq-Fw and pMopac12-seq-Rv primers,followed by a series of processes, including treatment with SfiIrestriction enzyme, ligation, and transformation, to constructsub-libraries in which the genes of the sorted cells were amplified. Atotal of 2 rounds of this procedure was performed. Thereafter, theresulting 40 clones were individually analyzed and an M428L variant withhigher affinity for FcRn at pH 5.8 than the wild-type Fc was sorted(FIG. 3 ).

Example 3: Construction of Error Library and Point Library of FcVariants

Two additional libraries were constructed using the sorted M428L as atemplate. First, mutations were introduced into Fc by error prone PCR toconstruct an error library. The library (size: 2×10⁸) was constructedusing ep-Fc-Fw and ep-Fc-Rv primers at such an error rate that 0.3%error (2.04 bp) was contained in Fc (680 bp). Second, M428L was used asa template to construct a point library. The library was constructedusing pMopac12-seq-Fw, pMopac12-seq-Rv, Fc-Sub #0-Rv, Fc-Sub #1-1-Fw,Fc-Sub #1-2-Fw, Fc-Sub #1-3-Fw, Fc-Sub #1-4-Fw, Fc-Sub #1-5-Fw, Fc-Sub#1-Rv, Fc-Sub #2-1-Fw, Fc-Sub #2-2-Fw, Fc-Sub #2-3-Fw, Fc-Sub #2-Rv,Fc-Sub #3-1-Fw, Fc-Sub #3-2-Fw, Fc-Sub #3-3-Fw, and Fc-Sub #3-4-Fwprimers such that mutations were randomly introduced into selectedregions where Fc were bound to FcRn (FIG. 4 ). Thereafter, a library ofFc variants was established by transformation into Jude1 in the samemanner as described above.

Example 4: Search Against the Error and Point Libraries of Fc VariantsBased on Bacterial Culture and Flow Cytometry and Sorting of Variants,Including PFc3, PFc29, PFc41, EFc29, EFc41, EFc82, and EFc88

The above sorting and resorting procedure was performed for theadditional error and point libraries constructed based on the sortedM428L. 5 rounds of sorting and resorting were repeated for the errorlibrary and only one round of sorting was performed for the pointlibrary. A group of about 100 clones from each of the two libraries wereindividually analyzed and Fc variants having high affinity for FcRn atpH 5.8 and low affinity for FcRn at pH 7.4 were sorted. FACS analysisrevealed that EFc6, EFc29, EFc41, EFc46, EFc70, EFc90 EFc82, and EFc88sorted from the error library showed higher fluorescence intensities atpH 5.8 than the wild-type Fc and conventional variants, including YTEfrom Medimmune (Gabriel J. Robbie et al., Antimicrob Agents Chemother.2013 December; 57(12): 6147-6153) and LS from Xencor (U.S. Pat. No.8,324,351). EFc6, EFc29, EFc41, EFc82, and EFc88 were found to showlower fluorescence intensities at pH 7.4 than LS. In addition, PFc3,PFc29, and PFc41 variants sorted from the point library showed higherfluorescence intensities at pH 5.8 than YTE and LS. PFc30 showed a lowerfluorescence intensity at pH 5.8 than YTE and LS. PFc29 and PFc41 showedlower fluorescence intensities at pH 7.4 than LS. Finally, EFc6, EFc29,EFc41, EFc82, EFc88, PFc3, PFc29, and PFc41 were selected because theyare expected to increase blood half-lives (Table 2 and FIG. 5 ).

TABLE 2 Name of Fc variant Positions of Fc variants and substitutedamino acids PFc 3 P228L/L309R/M428L/N434S (SEQ ID NO: 30) PFc 29Q311R/M428L (SEQ ID NO: 31) PFc 41 L309G/M428L (SEQ ID NO: 32) EFc 6P228L/V264M/L368Q/E388D/V422D/M428L/P445S (SEQ ID NO: 33) EFc 29P228L/R292L/T359A/S364G/M428L (SEQ ID NO: 34) EFc 41P228L/L234F/E269D/Q342L/E338D/T394A/M428L (SEQ ID NO: 35) EFc 82P230Q/F243Y/K246E/N361S/N384I/M428L (SEQ ID NO: 36) EFc 88P230S/Q295L/K320M/D356E/F405I/M428L (SEQ ID NO: 37) Point mutations ofthe sorted variants

The positions of the mutations are numbered according to the Kabat EUnumbering system, as described in Kabat et al., “Sequences of Proteinsof Immunological Interest”, 5th Ed., U.S. Department of Health and HumanServices, NIH Publication No. 91-3242, 1991).

Example 5: Production and Purification of Control Trastuzumab forIntroduction of the Fc Variants

Trastuzumab (Herceptin®), a representative IgG1 therapeutic antibody,was selected as a control group. In the subsequent examples, the sortedFc variants were introduced into trastuzumab.

The heavy and the light chain variable regions of wild-type trastuzumabwere synthesized (Genscript) from the corresponding amino acid sequencesfrom Drug Bank (http://www.drugbank.ca/) through mammalian codonoptimization simultaneously with back-translation. The synthesizedtrastuzumab heavy and light chain genes were sub-cloned into pOptiVEC-Fcand pDNA3.3 vectors, respectively (FIG. 6 ). Animal cell expressionplasmids encoding the trastuzumab heavy and light chains were prepared,expressed in HEK293 cells, and purified.

After culture in HEK 293F, the wild-type trastuzumab was purified byProtein A affinity chromatography (AKTA prime plus, cat #11001313) andgel permeation chromatography (HiTrap MabselectSure, GE, cat#11-0034-95). 7.7 mg of the wild-type trastuzumab was obtained in highpurity from 300 ml of the culture medium (FIG. 7 ).

Example 6: Analysis and Comparison of Physical Properties of In-HouseTrastuzumab and Commercial Trastuzumab

Unlike commercial trastuzumab produced by suspension culture in CHOcells, in-house trastuzumab was produced in HEK 293. Two basiccharacteristics of commercial trastuzumab and in-house trastuzumabantibodies were analyzed before introduction and function analysis ofthe sorted Fc variants. The pI values and charge variants of the sampleswere analyzed by capillary electrophoresis (CE: PA800 Plus, Beckmancoulter) using Pharmalyte 3-10 carrier ampholytes (GE Healthcare,17-0456-01) establishing a pH gradient of 3-10. The analytical resultsshowed that no impurities were detected by size exclusion chromatography(SEC, Tskgel G3000swxl, Tosoh). For the commercial trastuzumab, the pIvalues by charge variants were 8.27-8.74 and the pI of the main peak was8.62. For the in-house trastuzumab, the pI values by charge variantswere 8.29-8.78 and the pI of the main peak was 8.65, which were almostthe same as those for the commercial trastuzumab (FIGS. 8A1, 8A2 and8B). The pI values were measured by capillary electrophoresis (CE, PA800Plus, Beckman coulter). However, cIEF analysis revealed that there wereslight differences in the content of the in-house trastuzumab at themain peak and the other peaks. These differences are because of thedifferent glycan patterns of the in-house trastuzumab produced in HEK293cell line and the commercial trastuzumab produced in CHO cell line,leaving a possibility that the in-house trastuzumab might be oxidized bysialic acid. Thus, glycan analysis was also performed (FIGS. 9A and 9B).

After cleavage of N-glycan from the protein with PNGase F (NEB,186007990-1) and labeling with RapiFluor-MS reagent (Waters,186007989-1), glycan analysis was performed using a UPLC system (AcquityUPLC I class, Waters, FLR detector). As a result of the glycan analysis,the glycan patterns were similar but different glycan contents of thecompositions were observed, which seems to be not caused by sialicacid-induced oxidation but by the different production cell lines.Further, glycans were found top have no significant influence on bindingforce analysis and pharmacokinetic analysis (data not shown). Thus, thesorted Fc variants were introduced into the commercial trastuzumab andthe in-house trastuzumab.

Example 7: Production and Purification of the Fc Variants and Analysisof Physical Properties of the Fc Variants

Five control variants, including the commercial wild-type variant, thein-house wild-type variant, LS (XenCor), YTE (MedImmune), and 428L, andthe sorted variants PFc29, PFc41, EFc29, EFc41, and EFc82 weretransfected into HEK 293F animal cells. On the day before transfection,300 ml of HEK293F cells were passaged at a density of 1×10⁶ cells/ml. Onthe next day, cells were transfected with polyethylenimine (PEI,Polyscience, 23966). First, a heavy chain gene and a light chain gene ofeach of the variants were mixed in a 2:1 ratio in 30 ml of Freestyle 293expression culture medium (Gibco, 12338-018). Then, PEI and the variantgenes were mixed in a 1:2 ratio, left standing at room temperature for20 min, mixed with the cells that had been passaged on the previous day,cultured in a CO₂ shaking incubator at 125 rpm, 37° C., and 8% CO₂ for 6days, and centrifuged. The supernatant only was collected.

The proteins were purified from the supernatant by affinitychromatography using AKTA prime plus with a HiTrap MabselectSure column.300 ml of the supernatant was allowed to flow through the column at arate of 3 ml/min and washed with 100 ml of 1×PBS. Then, IgG Elutionbuffer (Thermo scientific, 21009) was allowed to flow through the columnat a rate of 5 ml/min. Six fractions (5 ml each) were collected. Eachfraction was neutralized with 500 μl of 1M Tris (pH 9.0). The fractionwas determined for proteins using Bradford (BioRad, 5000001) and put ina new tube. The purified variants were concentrated using a 30K Amiconultra centrifugal filter (UFC903096) and their physical properties wereanalyzed (FIGS. 10 and 11 ).

Each of the Fc variants other than the in-house wild-type trastuzumabwas purified with protein A and its purity (≥90%) and molecular weightwere determined by SDS-PAGE. SEC-HPLC (FIG. 11 a ) was used to obtainhigh-purity protein samples for efficacy evaluation and purity analysis(purity ≥97%). Analysis under isocratic conditions (mobile phase 1×PBS,pH 7.0, 1 ml/min flow rate) revealed that all Fc variants had the sameretention time for the main peaks and had estimated molecular weights(FIG. 11 b ).

Example 8: Measurement of Binding Forces of the Fc Variants to FcRn byELISA

ELISA was conducted to measure the pH-dependent binding forces of theprepared variants to FcRn and the binding forces of the variants toFcγRs and C1q, which allow the variants to exhibit effector functions.First, the pH-dependent binding forces of the variants to FcRn wereinvestigated. To this end, 50 μl of each of the IgG Fc variants dilutedto 4 μg/ml with 0.05 M Na₂CO₃ (pH 9.6) was immobilized onto aflat-bottom polystyrene high-bind 96-well microplate (costar) at 4° C.for 16 h, blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05%PBST pH 5.8/pH 7.4) at room temperature for 2 h, and washed four timeswith 180 μl of 0.05% PBST (pH 5.8/pH 7.4). Thereafter, 50 μl of FcRnserially diluted with 1% skim milk (in 0.05% PBST pH 5.8/pH 7.4) wasplated in each well and the reaction was carried out at room temperaturefor 1 h. After washing, an antibody reaction with 50 μl of anti-GST-HRPconjugate (GE Healthcare) was allowed to proceed at room temperature for1 h. The plate was washed and developed with 50 μl of 1-Step UltraTMB-ELISA Substrate Solution (Thermo Fisher Scientific). The reactionwas quenched with 2 M H₂SO₄ (50 μl each). Then, the reaction product wasanalyzed using an epoch microplate spectrophotometer (BioTek). Thesorted variants had binding forces to FcRn at pH 5.8 similar to thevariant LS and were more easily dissociated at pH 7.4 than LS (FIGS.12A-12D).

Example 9: Measurement and Comparison of Binding Forces of theTrastuzumab Fc Variants to Monomeric hFcRn at pH 6.0 and pH 7.4

In this example, the pH-dependent binding forces of the commercialtrastuzumab, the in-house trastuzumab, and the sorted Fc variants, whichwere analyzed and investigated for physical properties, to human FcRnwere compared. Specifically, K_(D) values were measured using a BiacoreT200 instrument (GE Healthcare). At pH 6.0, human FcRn was used as ananalyte in an antigen-mediated antibody capture format, as disclosed inthe literature (Yeung Y A. et al., J. Immunol, 2009). Each Fc variant asa ligand was diluted in running buffer (50 mM phosphate, pH 6.0, 150 mMNaCl, 0.005% surfactant P20, pH 6.0), injected at a level of ˜300response units (RUs) into the surface of CMS chip on which HER2 ECDdomain was immobilized to a level of ˜3,000 RUs, and captured. Forbinding force measurement, monomeric FcRn (Sinobiological inc.,CT009-H08H) as an analyte was serially diluted from 125 nM in FcRnrunning buffer, injected at a flow rate of 30 μl/min for 2 min, followedby dissociation for 2 min. In each cycle, regeneration was performedwith 10 mM glycine (pH 1.5) at a flow rate of 30 ml/min for 30 sec.Sensograms were fit to a 1:1 binding model using the BIAevaluationsoftware (Biacore). As a result, the Fc variants had higher bindingforces (PFc 3: 5.6 nM, PFc29: 6.8 nM, PFc 41: 5.9 nM, etc.) than thecommercial trastuzumab (15 nM) and the in-house trastuzumab (16.9 nM) ascontrol groups and 428L (9 nM) as the backbone. However, the Fc variantshad rather lower binding forces than YTE (5.7 nM) and LS (4.1 nM) whosebinding forces are known to be the highest values in the world, buttheir differences were almost the same within the error range. Since theligands were less bound to the analyte at pH 7.0, dissociation wasevaluated using an avid format in which monomeric hFcRn was directlyimmobilized and different concentrations of the Fc variants wereinjected (Zalevsky J et al. Nat. Biotechnol, 2010). Human FcRn ECDdomain (Sino Biological) was immobilized to a level of ˜1,500 RUs ontothe surface of a CMS chip. The Fc variants were serially diluted from3000 nM in HBS-EP (pH 7.4) and injected at a flow rate of 5 ml/min intothe FcRn-immobilized chip surface for 2 min. The bound Fc variants weredissociated for 2 min. After each cycle was finished, the chip surfacewas regenerated with 100 mM Tris (pH 9.0) (conatat time 30 sec; flowrate 30 μl/min). Particularly, the Fc variants PFc29 and PFc41maintained their high binding forces at pH 6.0 and were more rapidlydissociated at pH 7.4 than YTE and LS. These results were in agreementwith the results obtained by ELISA and suggest long expected half-livesof the Fa variants (FIGS. 13A and 13B). In practice, in vivopharmacokinetic experiments were conducted in human FcRn Tg mice.

Example 10: Analysis and Comparison of In Vivo PK Experiments of theCommercial Trastuzumab and the In-House Trastuzumab in Regular B6 Miceand hFcRn Tg Mice

PK analysis was conducted on regular B6 mice (Jungang ExperimentalAnimal Resource Center, C57BL/6J(B6)) whose genetic background isidentical to that of human FcRn Tg mice. As a result, the affinity ofthe Fc of a human antibody for regular mouse FcRn was found to be higherthan that for human FcRn, as reported in the literature. There was avariation in the PK values between the in-house antibody and thecommercial antibody in the regular mice, and the in-house antibodyappeared to be unstable. Further, the AUC in the Tg mice(B6.Cg-Fcgrttm1Dcr Prkdcscid Tg (Jackson lab, CAGFCGRT)276Dcr/DcrJ) wasslightly lower than that in the regular mice but the in-house antibodyand the commercial antibody showed similar pharmacokinetic tendencies.demonstrating that no problems were encountered in experiments using thein-house Fc variants produced in HEK293 to analyze the actual in vivopharmacokinetics of the Fc variants in the Tg mice (FIG. 14 ).

Example 11: Pharmacokinetics of Four Species, Including LS, YTE, andVariants PFc29 and PFc41 in hFcRn Tg Mice

The binding forces measured at pH 6.0 and pH 7.4 using an ELISA systemand a BiaCore instrument were found to be constant. Based on theseresults, PFc29 and PFc41 were sorted due to their high binding forcescomparable to that of LS under acidic conditions (pH 6.0) and higherdissociation forces at pH 7.4 than that of LS. Simultaneously with this,LS mutant from Xencor and YTE from MedImmune as control groups, whichare currently known to be most effective in the world, and the twotrastuzumab Fc variants were injected into 20 human FcRn Tg mice (5animals per group, 5 mg/kg I.V (tail vein)). After injection, bloodsamples were collected a total of 12 times (0, 30 min, 1 hr, 6 hr, 24hr, 3 day, 7 day, 14 day, 21 day, 28 day, 35 day, 42 day, and 50 day)from the facial vein. The concentrations of the Fc variants in the bloodsamples were analyzed by ELISA and then non-compartmental analysis (NCA)was conducted using WinNonlin. As expected from the results of ELISA andBiaCore analysis, the Fc variants PFc29 and PFc41 showed increased invivo half-lives. Particularly, the half-life of PFc29 was longer thanthat of conventional LS (FIG. 15 and Table 3).

TABLE 3 Trastuzumab Trastuzumab Parameter PFc29 PFc41 YTE LS (in-house)(commercial) t_(1/2) (day) 15.99 ± 9.57  10.87 ± 3.77 11.16 ± 7.21 11.72 ± 7.20  6.92 ± 1.06 6.57 ± 1.06 T_(max) (h) 0.70 ± 0.27  0.70 ±0.27 0.70 ± 0.27  0.70 ± 0.27  0.75 ± 0.27 0.79 ± 0.27 C₀ (μg/mL) 85.11± 11.31  69.72 ± 11.42 89.26 ± 12.54 82.72 ± 8.18 75.60 ± 4.11 64.98 ±40.08 C_(max) (μg/mL) 78.19 ± 9.55   65.22 ± 10.60 82.55 ± 11.36 75.92 ±7.07 70.40 ± 4.35 61.54 ± 9.29  AUC_(last) (μg/mL × day) 418.30 ± 120.22466.04 ± 51.57 520.26 ± 214.61  522.25 ± 182.53 249.60 ± 26.43 257.39 ±43.61  AUC_(inf) (μg/mL × day) 450.03 ± 158.08 489.92 ± 68.62 574.14 ±264.60  574.95 ± 228.28 250.84 ± 27.05 258.74 ± 44.32  AUC_(% Extrap)(%) 5.36 ± 6.20  4.55 ± 3.33 6.86 ± 6.57  7.24 ± 6.73  0.48 ± 0.24 0.49± 0.34 V_(z) (mL/kg) 292.54 ± 271.40 157.05 ± 41.29 126.70 ± 39.27 135.94 ± 52.09 158.91 ± 14.44 184.34 ± 22.30  CL (mL/day/kg) 12.29 ±4.31  10.36 ± 1.41 10.91 ± 5.96  10.21 ± 4.92  0.67 ± 0.07 0.83 ± 0.14Non-compartmental analysis of pharmacokinetic parameters of trastuzumabFc variants after intravenous administration (5 mg/kg) to mice (data areexpressed as mean ± SD (n = 5)). t_(1/2), terminal half-life; T_(max),time at maximal concentration; C₀, extrapolated zero time concentration;C_(max), maximal concentration; AUC_(last), area under the curve fromadministration to the last measured concentration; AUC_(inf), area underthe curve from administration to infinity; AUC_(% Extrap), percentage ofthe extrapolated area under the curve at the total area under the curve;V_(z), volume of distribution; CL, clearance.

Example 12: Measurement of Binding Forces of the Fc Variants to FcγRs byELISA

In this example, the binding forces of the Fc variants to FcγRs weremeasured. Specifically, 50 μl of each of the IgG Fc variants diluted to4 μg/ml with 0.05 M Na₂CO₃ (pH 9.6) was immobilized onto a flat-bottompolystyrene high-bind 96-well microplate (costar) at 4° C. for 16 h,blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 7.4)at room temperature for 2 h, and washed four times with 180 μl of 0.05%PBST (pH 7.4). Thereafter, 50 μl of FcγRs serially diluted with 1% skimmilk (in 0.05% PBST pH 7.4) was plated in each well and the reaction wascarried out at room temperature for 1 h. After washing, an antibodyreaction with 50 μl of anti-GST-HRP conjugate (GE Healthcare) wasallowed to proceed at room temperature for 1 h. The plate was washed anddeveloped with 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution(Thermo Fisher Scientific). The reaction was quenched with 2 M H₂SO₄ (50μl each). Then, the reaction product was analyzed using an epochmicroplate spectrophotometer (BioTek). Each experiment was conducted induplicate. FIGS. 16A thru 16L shows the binding forces of the Fcvariants to FcγRs (FcγRI, FcγRIIa(H), FcγRIIa(R), FcγRIIb, FcγRIIIa(V),and FcγRIIIa(F)), which were measured by ELISA.

Example 13: Measurement of Effector Functions of the Fc Variants byAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The antibody-dependent cellular cytotoxicity (ADCC) activities of thetrastuzumab Fc variants were evaluated using an ADCC reporter bioassaykit (Promega, G7010). Specifically, SKBR-3 cells as target cells wereplated at a density of 5×10³ cells/100 μl in each well of a 96-welltissue culture plate and cultured in a CO₂ incubator at 37° C. for 20 h.Thereafter, 95 μl of the culture medium was removed from each well ofthe plate using a multi-pipette and 25 μl of ADCC assay buffer providedfrom the ADCC reporter bioassay kit was plated in each well. Normal IgG,trastuzumab, and the trastuzumab Fc variants were diluted to variousconcentrations with ADCC assay buffer. 25 μl of each dilution was platedin each well of the 96-well tissue culture plate containing the cellsand left standing at room temperature until effector cells were added.Effector cells provided from the kit were dissolved in a thermostaticwater bath at 37° C. for 2-3 min and 630 μl of the solution was mixedwith 3.6 mL of ADCC assay buffer. 25 μl of the effector cells wereplated in each well of the plate containing the target cells and theantibody dilution. The reaction was carried out in a CO₂ incubator at37° C. for 6 h. After the lapse of a predetermined time, the plate wastaken out of the incubator and placed at room temperature for 15 min. 75μl of Bio-Glo™ Luciferase assay reagent was added to each well and thereaction was carried out at room temperature for 5 min. After completionof the reaction, the luminescence of each well was measured using aluminometer (Enspire multimode plate reader). The ADCC activity of eachtest antibody was determined by expressing the average of theexperimental results as a fold induction, which was calculated by thefollowing equation:

Fold induction=RLU(induced¹−background²)/RLU(no antibodycontrol³−background)

induced¹: RLU value acquired from the sample containing the targetcells, the test antibody and the effector cells

background²: RLU value acquired from the ADCC assay buffer

no antibody control³: RLU value acquired from the sample containing thetarget cells and the effector cells only

The ADCC activities of the trastuzumab Fc variants (LS, YTE, PFC29, andPFC41) for SKBR-3 were compared with that of trastuzumab (FIG. 17 ). Asa result, the maximum ADCC activities of LS, PFc29, and PFc41 at theirhighest concentrations were ˜1.5, ˜1.18, and ˜1.27-fold higher than thatof trastuzumab as the positive control group, respectively. In contrast,the maximum ADCC activity of YTE at its highest concentration was˜4.2-fold lower than that of trastuzumab. In conclusion, the trastuzumabFc variants PFc29 and PFc41 and the control variant LS achieved ADCCactivities 1.18-1.5 times higher than that of the control trastuzumab.The EC50 values of the variants were measured. As shown in FIG. 18 , thelower EC50 values of the Fc variant PFc29 (0.04543 μg/mL) and PFc41(0.05405 μg/mL) than the control LS (0.05575 μg/mL) indicate that theefficacies of the Fc variants PFc29 and PFc41 were more stable.

Example 14: Measurement of Binding Forces of the Fc Variants to C1q byELISA

In this example, the binding forces of the Fc variants to C1q weremeasured. Specifically, 50 μl of each of the IgG Fc variants diluted to4 μg/ml with 0.05 M Na₂CO₃ (pH 9.6) was immobilized onto a flat-bottompolystyrene high-bind 96-well microplate (costar) at 4° C. for 16 h,blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 7.4)at room temperature for 2 h, and washed four times with 180 μl of 0.05%PBST (pH 7.4). Thereafter, 50 μl of Complement C1q Human (Millipore)serially diluted with 1% skim milk (in 0.05% PBST pH 7.4) was plated ineach well and the reaction was carried out at room temperature for 1 h.After washing, an antibody reaction with 50 μl of anti-C1q-HRP conjugate(Invitrogen) was allowed to proceed at room temperature for 1 h. Theplate was washed and developed with 50 μl of 1-Step Ultra TMB-ELISASubstrate Solution (Thermo Fisher Scientific). The reaction was quenchedwith 2 M H₂SO₄ (50 μl each). Then, the reaction product was analyzedusing an epoch microplate spectrophotometer (BioTek). As a result of theanalysis, the binding force of the sorted PFc29 to C1q was higher thanthose of conventional LS and YTE (FIG. 19 ).

Although the particulars of the present invention have been described indetail, it will be obvious to those skilled in the art that suchparticulars are merely preferred embodiments and are not intended tolimit the scope of the present invention. Therefore, the substantialscope of the present invention is defined by the appended claims andtheir equivalents.

1. A polypeptide comprising a Fc variant wherein the Fc variant has anincreased half-life compared to a wild type, wherein the increasedhalf-life is by amino acid substitutions in the Fc domain of thewild-type, wherein the amino acid substitutions for the increasedhalf-life are M428L and Q311R or M428L and L309G according to the KabatEU numbering system.
 2. The polypeptide of claim 1, wherein the Fcvariant comprises a leucine at amino acid position 309 when the aminoacid substitutions of the Fc variant are M428L and Q311R.
 3. Thepolypeptide of claim 1, wherein the Fc variant has the amino acidsequence of SEQ ID NO:
 31. 4. The polypeptide of claim 1, wherein the Fcvariant has the amino acid sequence of SEQ ID NO:
 32. 5. The polypeptideof claim 1, wherein the polypeptide is a monoclonal antibody, abispecific antibody, an antibody conjugate, or a human antibody.
 6. Thepolypeptide of claim 1, wherein the polypeptide comprises a therapeuticprotein.
 7. The polypeptide of claim 6, wherein the polypeptide is a Fcfusion protein.
 8. A composition comprising the polypeptide according toclaim 1, a nucleic acid molecule encoding the polypeptide, or a vectorcomprising the nucleic acid molecule.
 9. The composition according toclaim 8, wherein the composition increases the blood half-life of anantibody or therapeutic protein for therapy in vivo.
 10. The compositionaccording to claim 9, wherein the composition is a pharmaceuticalcomposition for treating cancer.
 11. The composition according to claim10, wherein the antibody recognizes a cancer antigen.
 12. A nucleic acidmolecule encoding the polypeptide of claim
 1. 13. A vector comprisingthe nucleic acid molecule of claim
 12. 14. A host cell comprising thevector of claim 13.